Alma Mater Studiorum

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Alma Mater Studiorum Allma Mater Studiiorum – Uniiversiità dii Bollogna DOTTORATO DI RICERCA BIOTECNOLOGIE DEGLI ALIMENTI Ciclo XXI Settore scientifico disciplinare di afferenza: AGR/16 STUDIO DELLA MATURAZIONE DI FORMAGGI PECORINO STAGIONATI IN STABILIMENTO E IN GROTTA Presentata dalla dott.ssa: Elena Caffarri Coordinatore Dottorato Relatore Chiar.mo Prof. Chiar.ma Prof.ssa Giuseppe Losi Elisabetta Guerzoni Esame finale anno 2009 Alla mia famiglia Indice Parte introduttiva ................................................................ 1 Capitolo 1 Il formaggio ....................................................... 3 1.1 Le origini del formaggio ......................................................................... 3 1.2 Definizione e classificazione .................................................................. 5 1.3 Il formaggio Pecorino ............................................................................. 7 1.4 Tecnologia di produzione del Pecorino ................................................ 10 1.4.1 Preparazione del latte .................................................................... 11 1.4.2 Coagulazione del latte ................................................................... 12 1.4.3 Spurgo e rottura della cagliata ....................................................... 14 1.4.4 Cottura e Salatura .......................................................................... 15 1.4.5 Maturazione e Stagionatura........................................................... 15 Capitolo 2 Le G rotte di Santarcangelo ........................... 19 2.1 Cenni storici.......................................................................................... 19 2.2 La Geologia delle Grotte ...................................................................... 20 2.3 Le Grotte pubbliche .............................................................................. 21 2.4 La Grotta Teodorani ............................................................................. 22 Capitolo 3 Eventi legati alla maturazione ....................... 25 3.1 Maturazione nei formaggi .................................................................... 25 3.1.1 Enzimi coagulanti .......................................................................... 27 3.1.2 Proteinasi del latte ......................................................................... 30 3.1.3 Batteri acido lattici – starter .......................................................... 33 3.1.4 Batteri non-starter .......................................................................... 37 3.1.5 Catabolismo degli aminoacidi ....................................................... 38 3.1.6 Catabolismo degli aminoacidi solforati ........................................ 41 3.2 Lipolisi .................................................................................................. 43 3.2.1 Catabolismo degli acidi grassi liberi (FFA) .................................. 47 Capitolo 4 Eventi responsabili della formazione di aromi ............................................................................................. 51 4.1 Degradazione del lattosio ..................................................................... 51 4.2 Metabolismo del citrato ........................................................................ 53 4.3 Contributo della proteolisi allo sviluppo di aromi nel formaggio ........ 55 4.4 Contributo della lipolisi alla formazione di aromi ............................... 58 Capitolo 5 Ammine biogene ............................................. 61 I 5.1 Amine biogene negli alimenti .............................................................. 62 5.2 Tossicità delle ammine biogene ........................................................... 64 5.3 Microrganismi produttori di ammine biogene ..................................... 66 5.4 Limiti di legge ...................................................................................... 67 Capitolo 6 Microrganismi coinvolti nella maturazione del formaggio e fattori che ne influenzano la crescita .. 69 6.1 I batteri lattici ....................................................................................... 72 6.1.1 Lactobacillus ................................................................................. 73 6.1.2 Lactococcus .................................................................................. 73 6.1.3 Streptococcus ................................................................................ 73 6.1.4 Leuconostoc .................................................................................. 74 6.1.5 Pediococcus .................................................................................. 74 6.2 I clostridi .............................................................................................. 74 6.3 Pseudomonacee .................................................................................... 76 6.4 Bifidobatteri ......................................................................................... 76 6.5 Batteri propionici ................................................................................. 76 6.6 Lieviti ................................................................................................... 78 6.7 Muffe .................................................................................................... 79 6.8 Starter ................................................................................................... 80 6.9 Batteri lattici non-starter (NSLAB)...................................................... 82 6.10 Interazioni microbiche nel formaggio .............................................. 83 6.11 Biodiversità microbica nei sistemi caseari ....................................... 84 Capitolo 7 Analisi sensoriale ............................................ 85 7.1 L’analisi sensoriale dei formaggi e il modello Etana ........................... 86 Capitolo 8 Metodi per l’identificazione di microrganismi ............................................................................................. 91 8.1 Polymerase Chain Reaction (PCR) ...................................................... 91 8.2 ITS-PCR e RAPD-PCR ....................................................................... 93 Capitolo 9 Obiettivi .......................................................... 97 Capitolo 10 Materiali e metodi ...................................... 101 10.1 Determinazione dei parametri ambientali delle grotte ................... 101 10.2 Realizzazione della sperimentazione.............................................. 101 10.3 Analisi microbiologiche ................................................................. 101 10.3.1 Substrati utilizzati ....................................................................... 102 10.3.2 Ricerca di specie patogene.......................................................... 105 10.3.3 Isolamento e pre-identificazione dei microrganismi .................. 106 II 10.4 Determinazione del pH e dell’attività dell’acqua ........................... 107 10.5 Analisi del contenuto di ammine biogene ....................................... 107 10.5.1 Estrazione dei campioni (Pinho et al, 2001) ............................... 107 10.5.2 Derivatizzazione (Maijala e Eerola, 1993) .................................. 108 10.5.3 Condizioni cromatografiche ........................................................ 108 10.5.4 Preparazione degli standard ........................................................ 109 10.6 Caratterizzazione del profilo aromatico mediante analisi gascromatografica ......................................................................................... 110 10.7 Analisi di acidi grassi liberi ............................................................ 111 10.7.1 Estrazione .................................................................................... 111 10.7.2 Analisi gas-cromatografica degli acidi grassi estratti ................. 112 10.8 Valutazione organolettica dei prodotti ............................................ 112 10.9 Valutazione del processo di degradazione proteica ........................ 115 10.9.1 Trattamento dei campioni per l’ottenimento delle frazioni solubili (surnatante) e insolubili (pellet) ................................................................ 115 10.9.2 Elettroforesi su gel di poliacrilamide in presenza di sodio-dodecil solfato (SDS-PAGE) ................................................................................. 116 10.9.3 Elettroforesi su gel di poliacrilamide in presenza di Urea (Urea- PAGE) 119 10.10 Metodo di estrazione del DNA con CHELEX 5% per batteri Gram positivi (Walsh et al., 1991) .......................................................................... 121 10.11 Caratterizzazione genotipica mediante ITS-PCR ........................... 122 10.12 Sequenziamento del gene 16S-r-RNA ............................................ 123 10.13 PCR specie-specifica per Lactobacillus brevis (Guarnieri, Rossetti e Giraffa, 2001) ................................................................................................ 124 10.14 Multiplex PCR per Lactobacillus plantarum, Lactobacillus pentosus e Lactobacillus paraplantarum (Torriani, Felis e Dellaglio, 2001) ............... 124 Capitolo 11 Risultati e discussioni ................................. 129 11.1 Caratteristiche chimico-fisiche dei formaggi .................................. 129 11.2 Composizione microbica in rapporto all’ambiente di maturazione 131 11.3 Contenuto di ammine biogene
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